Abstract
The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.
Highlights
It has been proposed that the enzymes that are responsible for catalyzing sequential reactions in several metabolic pathways are highly organized in supramolecular complexes termed metabolons [1]
Tissue Distribution of BCAA Metabolon Protein Partners— Using rat liver mitochondrial extracts, we have shown previously the potential for a BCAA supramolecular complex consisting of BCATm and branched-chain ␣-keto acid dehydrogenase enzyme complex (BCKDC) proteins [5]
Other proteins were found in the liver mitochondrial complex. These non-BCKDC liver mitochondrial proteins, which bound to the BCATm affinity column, were GDH1, pyruvate carboxylase (PC), BDK, and NIPSNAP1 (Fig. 1)
Summary
It has been proposed that the enzymes that are responsible for catalyzing sequential reactions in several metabolic pathways are highly organized in supramolecular complexes termed metabolons [1]. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate. These results suggest that addition of PLP-BCATm plus a BCAA destabilizes the abortive complex of GDH1 by converting PLP-BCATm to the PMP form, releasing the BCKA product ␣-ketoacid.
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